Engineered antibody fragments with infinite affinity as reporter genes for PET imaging.
Reporter gene imaging has great potential for many clinical applications including the tracking of transplanted cells and monitoring of gene therapy. However, currently available reporter gene-reporter probe combinations have significant limitations with the biodistribution of the reporter probe and the specificity and immunogenicity of the reporter gene. The objective of the present study was to evaluate a new approach for reporter gene imaging based on cell surface expression of antibody fragments that can irreversibly bind to radiometal chelates.


METHODS
We developed a new reporter gene, designated 1,4,7,10-tetraazacyclodocecane-N,N',N'',N'''-tetraacetic acid (DOTA) antibody reporter 1 (DAbR1), which consists of the single-chain Fv (scFv) fragment of the anti-Y-DOTA antibody 2D12.5/G54C fused to the human T cell CD4 transmembrane domain. The corresponding reporter probe is yttrium-(S)-2-(4-acrylamidobenzyl)-DOTA (*Y-AABD), a DOTA complex that binds irreversibly to a cysteine residue in the 2D12.5/G54C antibody. U-87 glioma cells were stably transfected with a DAbR1 expression vector. Binding of *Y-AABD to transfected and wild-type cells was studied in vitro and in vivo.


RESULTS
Flow cytometry revealed high expression of the DAbR1 protein on the cell surface of tumor cells. Uptake of 90Y-AABD in DAbR1-expressing human U-87 glioma xenografts was 6.2 (+/-1.3) percentage injected dose per gram (%ID/g) at 1 h and 4.9 (+/-0.62) %ID/g at 24 h after injection. The corresponding tumor-to-plasma ratios were 45:1 and 428:1, respectively. Uptake by U-87 tumors without the DAbR1 gene was 0.16 (+/-0.02) %ID/g at 1 h and 0.05 (+/-0.03) %ID/g at 24 h. PET images in mice with 86Y-AABD demonstrated intense uptake in DAbR1-positive tumors and low background activity in the liver.


CONCLUSIONS
These findings indicate that cell surface expression of radiometal chelate binding antibodies such as 2D12.5/G54C is a promising strategy for reporter gene imaging.
Wei
Liu H
LH
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA.
Olafsen
Tove
T
Radu
Caius
C
Hildebrandt
Isabel J
IJ
McCoy
Mark R
MR
Phelps
Michael E
ME
Meares
Claude
C
Wu
Anna M
AM
Czernin
Johannes
J
Weber
Wolfgang A
WA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D045744:Cell Line, Tumor; D005240:Feasibility Studies; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics / Q000378:metabolism; D049268:Positron-Emission Tomography / Q000379:methods*; D015202:Protein Engineering; D013379:Substrate Specificity; D014018:Tissue Distribution; D015019:Yttrium / Q000737:chemistry
D000911:Antibodies, Monoclonal; D006573:Heterocyclic Compounds, 1-Ring; D007128:Immunoglobulin Fragments; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D015019:Yttrium